JP2005508839A - Antibodies against peptides and MUC1 protein - Google Patents

Abstract

  The present invention relates to methods for inhibiting proliferation or growth of tumor cells and / or inducing cell death in cancer cells. This method comprises the step of administering to a subject in need thereof an effective amount of a ligand that specifically binds to an epitope in the extracellular region of the transmembrane isoform of the MUC1 protein, whereby said method comprises It selectively inhibits the proliferation of or growth of mammalian cells. The present invention inhibits the growth or proliferation of epithelial tumor cells, colon tumor cells, lung tumor cells, breast tumor cells and ovarian tumor cells, or other cells expressing MUC1 protein, and induces the death of these cells For the use of antibodies, hybridomas, and pharmaceutical compositions containing the same.

Description

(Field of the Invention)
The present invention relates to a method of inhibiting tumor cell proliferation or growth and / or a method of inducing cell death in cancer. The present invention relates to antibodies, hybridomas and the like for inhibiting growth or proliferation and inducing death in epithelial, colon, lung, breast and ovarian tumor cells, or other cells expressing the MUC1 protein. It relates to the use of the pharmaceutical composition comprising.

(Background of the Invention)
Tumor markers are molecules that are involved in the transformation of normal cells to malignant tumor cells. Tumor markers are either mutated proteins that are different from those expressed in normal cells, or overexpression of proteins that are not or hardly expressed in normal cells.

  Mucins are high molecular weight glycoproteins that are produced by normal epithelial cells. MUC1 is one of the four mucins known to date, is a transmembrane molecule, and its function in adults is probably lubricious, but in the fetus it is located facing each other By keeping the cells separate, it is believed to perform an important function in forming the lumen of the tube. The MUC1 gene has also been shown to be expressed in hematopoietic tissues. Expression of MUC1 (or H23-Ag, episialin, PEM (polymorphic epithelial mucin), MCA (breast cancer tumor antigen) and EMA (epithelial cell membrane antigen)) is normal dormant lactating epithelial cells Are found to be elevated (10-50 times) in breast cancer cells. Furthermore, immunohistochemistry using H23 mAb (which recognizes MUC1) shows that MUC1 is expressed in 91% of breast cancer tissues and in 100% of breast cancer metastases, whereas non-malignant tumor tissues are H23 mAb. It was revealed that the staining was negative (1). Elevated levels of MUC1 protein in serum and body fluid have been reported in 7%, 17%, 64% and 67% of breast cancer patients with stages I-IV, respectively. Furthermore, elevated levels of circulating MUC1 may be associated with poor prognosis.

  It has been shown that MUC1 is overexpressed in epithelial cancers other than breast cancer. MUC1 has been shown to be overexpressed in epithelial ovarian cancer cells, as well as all types of lung cancer cells and other cancers (2).

  In malignant tumors, the MUC1 oligosaccharide chain is shorter and less dense compared to MUC1 in normal cells. This leads to the elucidation of novel epitopes of central proteins in cancer-associated mucins.

The isolation of MUC1 cDNA revealed several protein isoforms: MUC1 / REP protein, MUC1 / SEC protein, MUC1 / Y protein and MUC1 / X protein. A brief description of the four above-mentioned isoforms relevant to the present invention is provided below:
The MUC1 / REP isoform is a transmembrane protein, which is a large extracellular domain consisting of 20 amino acid repeat motifs that are heavily glycosylated (the number of these repeats varies from 20 to 100, thus VNTR (Named Variable Number of Tandem Repeat); has a transmembrane domain (consisting of a 28 amino acid hydrophobic sequence), and a 72 amino acid cytoplasmic domain. During the biosynthesis of the MUC1 / REP protein, it undergoes a proteolytic cleavage phenomenon. This cleavage occurs within the conserved sequence IKFRPGSVVV contained within the extracellular domain. Interestingly, this cleavage site residue of the previously identified modules is referred to as the “SEA” module (3), and is highly highly O which is uniformly bound to the cell membrane in one orientation or another. Found in proteins that are linked glycosylated. This SEA module functions not only as a site for proteolytic degradation, but also as a site for continuous reassociation of subunits. In conclusion, MUC1 / REP protein is a large cell bound by non-covalent and SDS-sensitive binding to smaller cell-fixing subunits (consisting of a small extracellular fragment followed by a transmembrane and cytoplasmic domain). Presented on the cell membrane as a heterodimer composed of outer subunits (including repetitive arrays). This large extracellular subunit can be cleaved and recombined with a small extracellular fragment of the cell-fixing subunit.

  The MUC1 / Y isoform is a transmembrane protein that contains the same transmembrane and cytoplasmic domains as the transmembrane and cytoplasmic domains of the MUC1 / REP protein. Unlike MUC1 / REP, the MUC1 / Y protein lacks both the tandem repeat array and its flanking regions due to differential splicing events that utilize splicing sites located upstream and downstream of the repeat array . Expression of MUC1 / Y has been demonstrated in various human secretory epithelial tumors. MUC1 / Y was found to be expressed on the cell surface of various human epithelial tumor cells but not detected in adjacent normal cells.

  MUC1 / X isoform (4) is a transmembrane protein that contains the same transmembrane domain and cytoplasmic domain as the transmembrane domain and cytoplasmic domain of the MUC1 / REP protein. Unlike MUC1 / REP, the MUC1 / X protein lacks both the tandem repeat array and its flanking regions due to differential splicing events that utilize splicing sites located upstream and downstream of the repeat array .

  MUC1 / SEC isoform: This isoform is generated by an alternative splicing mechanism. This protein is a secreted protein because it lacks a hydrophobic region capable of binding the protein to the cell membrane. This N-terminal sequence is identical to the sequence of the extracellular domain of MUC1 / REP. Furthermore, it has been shown that soluble secreted MUC1 / SEC protein can specifically bind to the extracellular domain of MUC1 / Y protein (5).

  Thus, for specific epitopes in the MUC1 protein, and more specifically, dramatically inhibits proliferation and growth and cell death of cells (eg, cancer cells and especially cells that overexpress the MUC1 protein). It is highly beneficial to develop ligands that bind to specific extracellular epitopes in the inducing MUC1 / REP protein or MUC / Y protein.

(Summary of the present invention)
In one embodiment, the present invention inhibits proliferation or growth and / or induces cell death in cancer cells that express MUC1 protein, and particularly in epithelial, breast, colon, lung and ovarian tumor cells. Provide a way to do that.

  In one embodiment, the present invention provides a method of selectively inhibiting cell proliferation and cell growth, and an effective amount that specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein Administering to the subject, thereby selectively inhibiting the proliferation or growth of such cells.

  In one embodiment, the present invention further provides a method of inducing cell death, wherein an effective amount of a ligand that specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein is provided to the subject. Administering and thereby selectively inducing cell death.

  In one embodiment, the present invention provides a method of selectively inhibiting cell proliferation and cell growth, and an effective amount of a ligand that specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein. And contacting cells expressing the MUC1 protein isoform, thereby selectively inhibiting cell proliferation or cell growth.

  The present invention provides a method for inducing cell death, contacting an effective amount of a ligand that specifically binds to an epitope in the extracellular region of the transmembrane isoform of the MUC1 protein and a cell expressing the MUC1 protein isoform. And thereby selectively inducing cell death.

  In one embodiment, the invention further provides a method of treating a subject afflicted with a disease associated with pathological proliferation of cells, epitopes in the extracellular region of the transmembrane isoform of MUC1 protein Administering an effective amount of a ligand that specifically binds to the subject, thereby treating the disease.

  In one embodiment, the invention further provides a method of treating a subject suffering from a disease associated with pathological proliferation of cells, wherein the extracellular region of the transmembrane isoform of MUC1 protein in the subject Administering an effective amount of the peptide to the subject, thereby treating the disease, to induce an increase in the level of antibodies specific for the peptide comprising an amino acid sequence that matches

  In one embodiment, the invention further provides an isolated antibody that specifically binds to an epitope in the extracellular region of the MUC1 protein isoform, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 1. In the 59 amino acid sequence.

  In one embodiment, the present invention further provides a pharmaceutical composition comprising an effective amount of a ligand that specifically binds to a MUC1 protein isoform, and a pharmaceutically acceptable carrier.

  In one embodiment, the invention further provides a hybridoma cell that produces a monoclonal antibody that specifically binds to an epitope in the extracellular region of the MUC1 protein isoform. In one embodiment, the epitope is located in a 59 amino acid sequence according to the amino acid sequence of SEQ ID NO: 1 and is located directly at the N 'end of the transmembrane domain of the protein.

(Detailed description of the invention)
MUC1 is one of four mucins that are transmembrane glycoproteins produced by normal epithelial cells. As previously described, MUC1 expression is markedly increased in all human breast cancer cells and other epithelial cancer cells. The sequence of MUC1 is shown in FIG.

  The term “specific binding” or “specifically binds” refers to an interaction between a protein, peptide, agonist, antibody or antagonist in the specification and claims section. This interaction depends on the presence of a specific structure of the protein (eg, an antigenic determinant or epitope) that is recognized by the binding molecule.

  In another embodiment, the ligand can be an antibody, antibody fragment, antagonist, agonist or peptide.

  The term “contacting a cell” as used herein and in the claims refers to administering a cell or exposing a cell to a ligand, which is a direct molecule known in the art. Depending on the method of application, it binds to a specific epitope in MUC1 / Y or MUC1 / X or MUC1 / REP. This ligand can be applied before or after seeding of the cells.

  The term “antagonist” refers to a molecule that, when bound to an epitope, reduces the amount or duration of the effect of the biological or immunological activity of the epitope. Antagonists can include proteins, nucleic acids, carbohydrates, antibodies, antisense, or any other molecule that reduces the effect of the MUC1 epitope on cell proliferation or cell viability.

“Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments. antibody).

  The term “agonist” as used herein and in the claims increases the amount or duration of the effect of the biological or immunological activity of an epitope when the epitope is bound or activated. Any molecule. The molecule can be a peptide construct or a nucleic acid construct.

  As demonstrated herein, Applicants have unexpectedly discovered the effect of antibody BOS7D10 on cell growth and / or cell proliferation clearly exemplified in Example 2 and FIG. The effect of this antibody and other antibodies on MUC1 / Y or MUC1 / SEC on cell viability was assessed by using MTT assays and microscopy, well known in the art. 59 amino acid sequence set forth in SEQ ID NO: 1, wherein the 59 amino acid sequence is located immediately from the transmembrane domain of the MUC1 / Y protein isoform and the MUC1 / X protein isoform and the MUC1 / REP protein isoform to the N-terminus. The effect of BOS7D10 on the epitopes contained in it was significant when compared to other antibodies that bind to different epitopes on the same protein.

  The term “selectively inhibiting cell proliferation or cell growth” as used herein and in the claims means death or growth (ie differentiation) or proliferation (ie Means that the pathologically proliferating cells are allowed to grow without leading to unacceptable death, or are propagated without reducing normal cell growth or proliferation), or reduced growth or proliferation To do. In one example, the death or reduction of growth (ie differentiation) or proliferation (ie propagation of cells that are pathologically proliferating) is at least 20%. The antibodies described in the present invention are shown as potential agents for inhibiting proliferation (FIG. 3A).

  “Selectively inducing cell death” refers to leading to at least a 20% reduction in pathological cell viability in the present specification and claims. Cell death assesses cell viability (eg, morphology, or use of different dyes such as MTT assay or propidium iodide (PI) staining), trypan blue assay, pre-cytotic DNA It can be demonstrated by methods of fragmentation, changes in membrane symmetry, activation of apoptotic caspases, and release of cytochrome C into the cytoplasm by mitochondria, degradation of actin filaments well known to those skilled in the art.

  There can be many processes to achieve cell death, and some of these can induce apoptosis. Although there are other cell death mechanisms other than apoptosis, there are speculations that there are several features of cell death that are unrelated to the way cells reach cell death. One of these cell death features is the absence of metabolism, and another is enzymatic denaturation. In either case, viable strains cannot stain these cells. These endpoints of cell death have long been understood and expedite the current understanding of cell death mechanisms. Furthermore, there is a difference between a cytotoxic effect in which cells are killed and a cytostatic effect in which cell proliferation is inhibited. In accordance with the data presented in the Examples section, the antibodies of the invention are effective as cytotoxic and cytostatic agents and are considered apoptotic antibodies.

  A “cell” of the present invention is a pathologically proliferating cell, such as a cancer cell that expresses a MUC / 1 protein, according to another embodiment of the present invention in the specification and claims section. A cell. In another embodiment, the MUC1 protein is overexpressed.

  The cancer cells can be, but are not limited to, epithelial cells, breast cancer cells, colon cancer cells, lung cancer cells, or all cells that express the MUC1 protein isoform. MUC1 protein isoforms include, but are not limited to, protein isoforms such as MUC1 / Y protein, MUC1 / X protein, MUC1 / REP protein and MUC1 / SEC protein. “Isoforms” are translated from the same gene but may contain different numbers of amino acids and / or different additional groups and / or have different molecular structures due to post-translational or post-transcriptional modifications It is a protein.

  MUC1 / Y, MUC1 / X and MUC1 / REP have identical transmembrane and cytoplasmic domains. More importantly, the domain located from the transmembrane domain to the N 'terminus is identical in these two isoforms (red box in Figure 2). Thus, the antibodies of the invention can bind to an epitope present on both protein isoforms.

The term “epitope” refers to a specific portion of an antigen that binds to a specific antibody. According to an embodiment of the invention, the length of the epitope is 4-12 amino acids. In another embodiment, the epitope length is 5-10 amino acids, and in yet another embodiment, the epitope length is 6-8 amino acids. The sequence of the epitope is contained in SEQ ID NO: 1 and the 59 amino acid sequence shown below:
(N-terminus) SVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPPFPFSA QSGAGV (C-terminus).

  In another embodiment, the epitope is located in a 15 amino acid sequence present in the N-terminal portion of a 59 amino acid segment located directly at the N-terminus of the transmembrane domain of MUC1 / Y protein, MUC1 / X protein and MUC1 / REP protein. It is done. The epitope is located in the extracellular region of the transmembrane isoform of MUC1 / Y protein, MUC1 / X protein and MUC1 / REP protein.

  The term “extracellular region” as used herein and in the claims refers to a stretch of various numbers of amino acids that are located immediately from the transmembrane domain to the N-terminus and appear to be present in the extracellular environment.

  The term “transmembrane” is used herein and in the claims to refer to amino acids that are predominantly hydrophobic, located within the membrane, from the cytoplasmic domain of the protein to the N-terminus and from the extracellular domain of the protein to the C-terminus. Is a stretch. This domain allows the protein to be anchored to the membrane.

  The term “antibody”, as used herein and in the claims, refers to an immunoglobulin that occurs naturally or is partially or wholly synthetically produced. The antibody can be a human antibody or an animal antibody. The term also encompasses any polypeptide or protein that has a binding domain that is or is homologous to an antibody binding domain. These antibodies can be derived from natural sources, or can be made partially or wholly synthetic. Examples of antibodies can be immunoglobulin isotypes and subclasses of those isotypes; fragments (which contain antigen binding domains such as Fab, scFv, Fv, dAb, Fd); and diabodies. As illustrated in Example 2, antibody BOS7D10 showed a potential inhibitory effect on cell proliferation and a potential cell death activity on MCF7.

  Recombinant DNA engineering techniques can be used to generate other antibodies or chimeric molecules that require monoclonal and other polyclonal antibodies and maintain the specificity of the original antibody. Such techniques include introducing a constant region, or a DNA that encodes an immunoglobulin variable region, or complementarity determining region (CDR) of an antibody to a constant region plus a framework region of a different immunoglobulin. . See, for example, EP-A-184187, GE2188638A or EP-A-239400. Since an antibody can be modified by a number of methods, the term “antibody” should be constructed to cover any specific binding member or substance having a binding domain with the required specificity. Thus, the term includes antibody fragments, derivatives, functional equivalents and homologs of this antibody (any polypeptide comprising an immunoglobulin binding domain, whether natural, wholly or partially synthetic). Cover). Thus, chimeric molecules comprising an immunoglobulin binding domain, or equivalents, fused to other polypeptides are included. Cloning and expression of chimeric antibodies are described in EP-A-120694 and EP-A-0125023.

  It has been shown that fragments of whole antibodies can perform the function of the bound antigen. Examples of binding fragments are: (i) Fab fragment consisting of VL domain, VH domain, CL domain and CHI domain; (ii) Fd fragment consisting of VH domain and CHI domain; (iii) VL domain and VH domain of a single antibody (Iv) a dAb fragment consisting of a VH domain (8); (v) an isolated CDR region; (vi) an F (ab ′) 2 fragment comprising two linked Fab fragments A bivalent fragment (vii) single chain Fv molecule (scFv), where the VH and VL domains are linked by a peptide linker that joins the two domains to form an antigen binding site (9-10) (Viii) Bispecific (bisspecif) c) single chain Fv dimers (PCT / US92 / 09965) as well as (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; (11)) A diabody is a multimer of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain; The two domains are linked (eg, by a peptide linker) but cannot associate with each other to form an antigen binding site: the antigen binding site is a first domain of a polypeptide within a multimer and this Formed by binding to a second domain of another polypeptide within the multimer (W 94/13804).

  One of the potential advantages of monoclonal antibodies with respect to cancer treatment is their ability to specifically recognize a single antigen. In some cases, cancer cells were thought to have antigens specific for the transformed cell species. Currently, cancer cells have few unique antigens; rather, these cancer cells tend to overexpress normal antigens (eg, MUC1 protein) or to express fetal antigens. Seems frequent. Nevertheless, the use of monoclonal antibodies provides a method for delivering a dose of a reproducible antibody to a patient who is expected to have a better response rate than a polyclonal antibody.

  As illustrated in Example 3, a surprising and unexpected correlation was found between the effect of the antibody on the interaction of MUC1 / SEC and MUC1 / Y and the effect of this antibody on cell death. ing. Possible antibodies in inducing cell death were also possible in inducing isotype tube interactions, and vice versa (see Table 1). This surprising correlation can be related to the mechanism of action of the antibody in inducing cell death.

  The present invention further provides an isolated antibody that binds to the described epitope. The antibody can be a monoclonal antibody, a synthetic antibody, a polyclonal antibody, or a chimera (eg, a murine variable region bound to a human constant region).

  In another embodiment, the present invention provides a method of treating a subject having a disease associated with pathological cell proliferation, which method corresponds to the extracellular region of the transmembrane isoform of MUC1 protein. Administering an effective amount of a peptide comprising an amino acid sequence to a patient in need, eg, the 59 amino acid sequence of SEQ ID NO: 1, which is the N ′ of the transmembrane domain of the MUC1 protein. Directly located at the end. In accordance with the present invention, the peptide induces increased levels of antibodies specific for the peptide in a subject. The term “inducing an increase in the level of an antibody specific for a peptide” refers to an increase in the level of a specific antibody directed against this peptide that exceeds the basal amount of this antibody in an untreated subject. Say.

  The amino acid sequence of the region that induces the epitope of the antibody is shown in FIG. Based on the registration number ATCC-registration number XXXX, BOS7D10 is based on American Type Culture Collection, Rockville, Md. It is deposited with the USA.

  The term “amino acid” or “amino acid sequence” refers to an oligopeptide, peptide, polypeptide, or protein sequence, or any fragment thereof, and refers to a naturally occurring or synthetic molecule.

  The invention further provides a pharmaceutical composition comprising an amount of a ligand (eg, an antibody, peptide or antagonist that specifically binds to the MUC1 protein) and a pharmaceutically acceptable carrier.

  The term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

  The invention can be further expanded to treat mammals (especially humans) having a disease associated with pathological mammalian cell proliferation (eg, cancer). “Treat” means: (i) Preventing a disease (this disease may occur in an animal that is likely to have the disease, disorder, and / or condition but has not yet been diagnosed to have it) (Ii) inhibiting the disease (ie, preventing its development); and (iii) reducing the disease (ie, causing regression of the disease).

  Accordingly, the present invention further provides a method of inhibiting pathological growth or cell growth of mammalian cells, which specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein. Administering an effective amount of a ligand to a subject in need thereof, thereby selectively inhibiting abnormal growth or proliferation of mammalian cells.

  In another embodiment, the present invention provides a method of inducing cell death comprising: an effective amount of a ligand that specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein. Administering to a subject in need thereof, thereby inducing cell death.

  The administering step may involve direct injection of either ligand with a pharmaceutically acceptable carrier targeted to the target cells. Alternatively, prodrugs that are activated only in the required target cells, either by different enzymes or by different chemical-physical conditions, can be used.

  Liposomes have been used to successfully administer drugs to cancer patients and are clinically useful in the delivery of anticancer drugs (eg, doxorubicin conjugates, daunorubicin conjugates and cisplatinum conjugates) Is shown (12-14).

  Similarly, micelles have also been used to deliver drugs to patients (15), and micelles have been used as drug carriers and for targeted drug delivery (16-17), Contains cancer drugs (18-19).

  Where the antibody or composition is suitable for oral administration, the formulation may contain, in addition to the active ingredient, additives such as the following: starch (eg, potato starch, corn starch or wheat starch) or cellulose derivative Or starch derivatives (eg microcrystalline cellulose); silica; various sugars (eg lactose); magnesium carbonate and / or calcium phosphate. Where the oral formulation is for administration, it is desirable that it be well tolerated by the patient's digestive tract. In order to achieve this goal, it may be desirable to include a mucus formr and a resin. It is also desirable to improve tolerance by formulating the antibody or composition in capsules that are insoluble in gastric juice. It may also be preferred to include the antibody or composition in a sustained release formulation.

  Where the antibody or composition is suitable for rectal administration, the formulation comprises a binder and / or lubricant (eg, a polymeric glycol, gelatin, cocoa butter or other vegetable wax or vegetable fat). obtain.

  The pharmaceutical compositions utilized in the present invention may be administered by any number of routes including, but not limited to: oral means, intravenous means, intramuscular means, intraarterial means, intramedullary means, arachnoid means Intracavitary means, intraventricular means, transdermal means, subcutaneous means, intraperitoneal means, intranasal means, enteral means, topical means, sublingual means or rectal means. Alternatively, the antibody or composition can be in dry form for reconstitution with a suitable sterile liquid prior to use.

  In addition to the active ingredient, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers, including excipients and adjuvants, which facilitate the processing of the active compound into preparations. This can be used pharmaceutically. Further details on techniques for formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton Pa).

  Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such carriers allow the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for ingestion by the patient. .

  Pharmaceutical preparations for oral use combine the active compound with solid excipients to obtain tablets or dragee cores and process the mixture of the resulting granules (if necessary after milling) Can be obtained through Appropriate adjuvants can be added if desired. Suitable excipients include carbohydrate or protein fillers (eg, sugars (lactose, sucrose, mannitol, and sorbitol); starch from corn, wheat, rice, potato or other plants; cellulose (eg, methylcellulose, hydroxy Propylmethylcellulose, or sodium carboxymethylcellulose), gums (including gum arabic and tragacanth), and proteins (eg, gelatin and collagen) If desired, degrading or solubilizing agents (eg, cross-linked polyvinyl pyrrolidone). , Agar and alginic acid, or salts thereof (eg, sodium alginate)) can be added.

  Dragee cores can be used with suitable coating agents such as concentrated sugar solutions, which can also include gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions As well as a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragees for product identification or to characterize the quantity of active compound (ie, dosage).

  Pharmaceutical preparations (which can be used orally) include pressure-fit capsules made from gelatin, as well as soft, sealed capsules made from gelatin and a coating, such as glycerol or sorbitol Pressure-compatible capsules include active ingredients mixed with fillers or binders (eg lactose or starch), lubricants (eg talc or magnesium stearate), and optionally stabilizers. obtain. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids such as fatty oils, liquids, liquid polyethylene glycols with or without stabilizers.

  Pharmaceutical formulations suitable for parenteral administration are formulated in aqueous solutions, preferably in physiologically compatible buffers (Hank's solution, Ringer's solution, or physiologically buffered saline). Can be done. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable affinity fatty solvents or vehicles include fatty oils (eg, sesame oil), or synthetic fatty acid esters (eg, ethyl oleate), triglycerides, or liposomes. Non-lipid polycationic amino polymers can be used for delivery. If desired, the suspension may also contain suitable stabilizers or agents that enhance the solubility of the compound and make it possible to prepare highly concentrated solutions.

  For topical or intranasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

  The pharmaceutical compositions of the present invention are in a manner known in the art (eg, by conventional mixing, dissolving, granulating, dragee making, pasting, emulsifying, encapsulating, incorporating, or lyophilizing process). Can be manufactured.

  After the pharmaceutical compositions are prepared, these compositions can be placed in a suitable container and labeled for treatment of the indicated conditions.

  Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredient is included in an amount effective to achieve its intended purpose. The determination of an effective dose is well within the ability of those skilled in the art.

  For any compound, the therapeutically effective dose can be estimated initially either in cellular assays for neoplastic cells or in animal models (eg, in mice, rats, rabbits, dogs, or pigs). The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine effective doses and routes for administration in humans. Effective doses are also determined in clinical trials in humans.

  The ligand of the present invention can promote the interaction between MUC1 / Y and MUC1 / SEC. Morphological changes are observed in FIGS. 3B-D and may be involved in the interaction of these two isoforms, where MUC1 / SEC functions as a ligand for MUC1 / Y acting as a receptor.

  The term “cancer” is interpreted broadly. A ligand (eg, antibody or antagonist) of the invention can be an “anticancer agent”, which term can also include an “anti-tumor cell proliferating agent” and an “anti-neoplastic agent”. For example, the methods of the invention are useful for the treatment of cancer by inducing cell death or inhibiting the proliferation / or growth of pathological cells.

  The invention further provides a hybridoma cell that produces a monoclonal antibody that binds to an epitope in the extracellular region of the isoform of the MUC1 protein.

  Hybridomas producing the monoclonal antibodies of the invention are generated according to the general procedure described by Kohler and Milstein, Nature, 256, p495 (1975). In this procedure, the hybridoma uses somatic cell hybridization procedures to fuse antibody-producing cells (typically mouse spleen cells previously immunized with a mucin antigen source) with an immortalized tumor. To be prepared. The drug was used for immunization of animals (“immunogen”) to induce the production of antibodies against mucin antigens.

  For the production of antibodies, various hosts (including goats, rabbits, rats, mice, humans and others) inject the relevant epitope, or any fragment or oligoepitope thereof having immunogenic properties. Can be immunized. Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include Freund, mineral gels (eg, aluminum hydroxide) and surfactants (eg, lysolectin, pluronic polyols, poranions, peptides, oil emulsions, KLH, and dinitrophenol). ), But is not limited thereto. Among the adjuvants used in humans, BCG (Calmettgeran) and Corynebacterium parvum are particularly preferred Freund adjuvants.

  The hybridoma resulting from the fusion process is allowed to grow. The resulting supernatant is then screened using an immunoassay procedure to detect antibodies present in the supernatant that can bind to a specific antigen. In other cases, the supernatant was screened for the ability to bind to cultured cancer cells.

  The antibody is designed for therapeutic treatment of cancer in a patient. Thus, the antibody can be a naked antibody. In another embodiment, the antibody can be conjugated to a cytotoxic drug. The term cytotoxic drug refers to any agent that kills cells containing, but not limited to, for example, radioisotopes. Antibodies can be used to target other molecules to cancer cells. Examples of cytotoxic drugs include, but are not limited to, bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, and cisplatin.

  It is understood that the invention is not limited by what has been described, and it is understood that there are numerous modifications, all of which are within the scope of the invention. For example, although the invention has been described with respect to tested antibodies, other antibodies that overlap portions of the described epitopes can demonstrate similar effects.

  It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove.

(Example)
(Materials and methods)
(Cell line)
The HBL-100- epithelial cell line was obtained from a primary culture of cells derived from early milk of human milk (20). The cells contain SV40 incorporated in tandem.

  HBL / neo (H / N) —appropriate transfectants were generated by transfecting the cloning vector pSV2neo into HBL-100 cells.

  HBL / Y2 (H / Y2) -appropriate transfectants were generated by co-transfecting the expression plasmid carrying MUC1 / Y cDNA with the cloning vector pSV2neo.

(Medium for cell culture growth)
DMEM growth medium (Dulbecco's modified Eagle medium) was purchased from Biological Industries, Kibbutz Beit Haemek, Israel.

(Medium supplement :)
Antibiotic: 10 μg / ml nystatin, 100 μg / ml streptomycin, 100 μg / ml ampicillin (Biological industries, Kibbutz Beit Haemek, Israel).

  4 mM L-glutamine (Biological industries, Kibbutz Beit Haemek, Israel).

  Heat inactivation (56 ° C., 30 min) 10% fetal calf serum (FCS) (Biological industries, Kibbutz Beit Haemek, Israel).

  Mixed oxaloacetate (Sigma), pyruvate (Sigma), insulin (Sigma) (OPI) was added to the hybridoma growth medium.

  (Hybridoma SM (serum free) medium) -DMEM growth medium (Dulbecco's modified Eagle medium) was diluted 1: 1 with F12 medium (Biological industries, Kibutz Beit Haemek, Israel) and antibiotics (10 μg) / Ml nystatin, 100 μg / ml streptomycin, 100 μg / ml ampicillin), 4 mM L-glutamine and “BIOGRO-2” (synthetic serum-free medium supplement). All of the above materials were purchased from Biological Industries, Kibbutz Beit Haemek, Israel.

  (Flow cytometry medium) -DMEM was supplemented with the above antibiotics, 5% FCS and 0.01% Na-Azide.

(antibody)
H23—a monoclonal antibody that recognizes epitopes in the MUC1 tandem repeat array, thereby detecting MUC1 / REP and MUC1 / SEC.

  BOS6E6-An anti-MUC1 / Y-specific monoclonal antibody that recognizes a MUC1 / Y-specific epitope.

  BOS7D10, BOS10D2, BOS10B3- A monoclonal antibody that recognizes an epitope in the MUC1 / Y extracellular domain.

  SEC7H10—An anti-MUC1 / SEC specific mAb that recognizes the 11 amino acids at the C-terminus of MUC1 / SEC.

(Cell culture)
(Proliferation of cells in culture)
Cells were cultured in 25 cm ² or 75 cm ² tissue culture flasks (Falcon Costar) with 5 ml or 10 ml culture medium, respectively, and kept in an incubator at 37 ° C. with 95% air and 5% CO ₂ . .

(Cell collection and inoculation)
Collection of cells growing in the monolayer was performed by adding 0.5-1 ml trypsin-EDTA solution to the flask for 1-5 minutes (cell type selection). Next, an equal volume of 10% FCS rich growth medium was added to neutralize enzyme activity. Cells were inoculated into tissue culture flasks containing growth media.

(Cell count)
Cells were counted using a hemocytometer. To detect dead cells, the cells were resuspended in medium containing 0.08% trypan blue.

(Cell transfection)
Transfection of cells was performed using the following commercially available transfection kits: DOTAP transfection reagent kit (Roche diagnostics), Effectene Transfection Reagent (Qiagen) and LipofectAMINE Transfection reagent (Gibco-BRL). The transfection protocol was performed according to the manufacturer's manual.

(Flow cytometry analysis (FACS analysis))
Cell samples (10 ⁶ cells / sample) were washed with flow cytometry medium and incubated for 2 hours at 4 ° C. with primary antibody diluted in flow cytometry medium. Cells are washed with flow cytometry medium and incubated with secondary anti-mouse IFTC-conjugated antibody (diluted 1:50 with flow cytometry medium) at 4 ° C. for 45 minutes, followed by extensive washing, Cells were then resuspended in PBS containing 0.01% Na-azide. The sample was analyzed by a FACS analyzer (Beckton Dickenson). The flow cytometry results were analyzed using “Cell Quest” software.

(MTT proliferation assay)
20 μl of 0.5% MTT (Sigma) solution (in phosphate saline buffer) was added to HBL-Y cells growing in 200 μl medium in a 96 well plate. The cells were incubated with MTT reagent for 2 hours at 37 ° C., the medium was removed from the cells, and MTT crystals were lysed in 95% ethanol for 15 minutes, and the plate was read by an ELISA reader at 592 nm.

(MUC1 / SEC-MUC1 / Y interaction ELISA assay 96 microtiter)
“Maxi Sorp” plates (NUNC) were coated overnight at room temperature with 100 μl conditioned medium containing MUC1 / SEC. The following coated plates were washed twice with phosphate saline wash buffer (pH 7.0 (neutralized with 1M HCl)) containing 0.05% Tween 20 (0.05%) Then, blocking was performed at 35 ° C. for 2 hours with a phosphate physiological saline washing buffer supplemented with 10% FCS. Recombinant MUC1 / Y diluted in phosphate saline wash buffer supplemented with 10% FCS was incubated in the plate at 35 ° C. for 2 hours. Following incubation, the plates were washed extensively with phosphate saline wash buffer and further at 35 ° C. with biotinylated 6E6 / 2 mAb diluted 1: 300 in phosphate saline wash buffer. Incubate for 2 hours followed by extensive washing with phosphate saline wash buffer and incubate with streptavidin-HRP (diluted 1: 1000). This reaction was developed using OPD substrate (Sigma). The reaction was stopped by the addition of 30 μl 2M sulfonic acid and read at 490 nm.

(vector)
pSV2neo-a vector encoding a neomycin resistance gene that allows selection of stably transfected eukaryotic cells using G418 (21).

  pCL642 encoding the pCL642-MUC1 / Y-MUC1 / Y isoform (6).

(DNA ligation)
The reaction was carried out overnight by incubating the following mixture while gradually decreasing the temperature from 25C to 4C.

（プラスミド調製）
小スケールおよび大スケールの両方でのプラスミド調製（それぞれＭｉｎｉ ｐｒｅｐおよびＭａｘｉ ｐｒｅｐ）を、Ｍａｎｉａｔｉｓハンドブック（Ｓｍｂｒｏｏｋら、１９９０）に従って実行した。

(Plasmid preparation)
Plasmid preparation at both small and large scale (Mini prep and Maxi prep, respectively) was performed according to the Maniatis handbook (Smbrook et al., 1990).

DNA concentration was determined by converting absorbance (OD) units at 260 nm according to the following formula: 1O. D _260nm = 40μg
Example 1
(Establishment of MUC1 / Y expressing cells)
In order to generate MUC1 / Y expressing cells, the epithelial cell line HBL-100 was co-transfected with a eukaryotic expression vector containing MUC1 / Y cDNA and the selection plasmid pSV2neo. Control cells were generated by transfecting cells only with a plasmid conferring neomycin resistance. After transfection, a number of control transfectants (H / N) were generated and a single clone (H / Y2) that consistently expressed the MUC1 / Y protein was generated. Expression was confirmed by flow cytometric analysis using anti-MUCl / Y mAb (Figure 1).

  As can be observed from the flow cytometric analysis described above, both H / Y2 and H / N transfectants express endogenous MUC1 tandem repeat arrays containing isoforms.

(Example 2)
(Effect of monoclonal antibody recognizing MUC1 / Y protein and MUC1 / SEC protein on epithelial cell viability)
A number of antibodies against the MUC1 protein have been used. Its immunoreactive region within the MUC1 protein has been defined (FIG. 2).

  To evaluate the effect of anti-MUC1 / Y and MUC1 / SEC monoclonal antibodies on human epithelial cell proliferation, MUC1 / Y-expressing human epithelial cell transfectants (H / Y2) were treated with anti-MUC1 / Y (MUC1 / Y Wells pre-coated with serum-free hybridoma supernatants containing either 4 mAbs) that recognize different epitopes in the extracellular domain) or anti-MUC1 / SEC monoclonal antibodies.

  A dramatic effect was observed when cells were plated into wells coated with monoclonal antibody BOS7D10 (FIG. 3A, rows 2 and 2 'and panel C). The other three anti-MUCl / Y monoclonal antibodies had no discernable effect (Figure 3A, columns 1 and 1 ', 3 and 3', 4 and 4 ', and panels B and C). The potent inhibitory effect of the hybridoma BOS7D10 was further observed according to an 8-fold dilution of the serum-free hybridoma supernatant and the titration was terminated at the final 16-fold dilution.

  Similar results were observed when the antibodies were tested on MCF7 human and breast cancer cells as target cells. In this case, the monoclonal antibody BOS7D10 also exerted a strong cell death activity in MCF7 cells. Other antibodies had no significant effect on the cells.

  As demonstrated in FIG. 3D, microscopic analysis revealed that cells treated with other antibodies and cells treated with BOS7D10 had different appearances (FIGS. 3B, C). In fact, they show the morphology of non-viable cells.

  “Box” titration of the BOS7D10 hybridoma supernatant (FIG. 4) against fetal calf serum concentration showed that the cells grew at a final serum concentration of 0.3% and to a lesser extent 1% Cell growth inhibitory activity can be clearly observed when discernable by final serum concentration (FIG. 4). Cell growth at serum concentrations higher than 1% abrogated the growth inhibitory activity exerted by the BOS7D10 antibody.

  Interestingly, the monoclonal antibody SEC7H10 against the 11 C-terminal amino acids of the MUC1 / SEC protein also demonstrated growth inhibitory activity (FIG. 3A, rows 5 and 5 '). However, this growth inhibition was significantly less than exhibited by BOS7D10. Control monoclonal antibody isoforms consistent with BOS7D10 and SICH10 antibodies produced any effect (data not shown).

Example 3
(Effect of anti-MUC1 / Y antibody and anti-MUC1 / SEC antibody on the binding of MUC1 / Y and MUC1 / SEC)
In parallel with the effects of anti-MUC1 / Y and anti-MUC1 / SEC antibodies on epithelial cell proliferation, we can more or less affect the binding of MUC1 / SEC protein to MUC / Y protein. (See method for assay description).

  The antibody BOS7D10, which recognizes an epitope in the extracellular domain of MUC1 / Y, markedly enhanced MUC1 / Y binding to MUC1 / SEC protein. The anti-MUCl / SEC antibody SEC7H10 also increased binding slightly but less than BOS7D10 (see Table 1). Other antibodies do not affect the MUC1 / Y MUC1 / SEC interaction in the same way (Table 1). Surprisingly, these results correlate directly with antibodies that affect cell proliferation. BOS7D10, which induces maximum cell growth inhibition, also has a strong effect on the MUC1 / Y MUC1 / SEC interaction. Similar correlation was shown for SEC7H10. FIG. 5 shows that antibodies that increase MUC1 / Y-MUC1 / SEC binding can induce a negative proliferation signal. This result is in good agreement with previous data suggesting that MUC1 / SEC binding to MUC1 / Y may exert a growth inhibitory signal. Indeed, the addition of conditioned medium containing MUC1 / SEC protein to cells expressing MUC1 / Y protein reduces the growth rate of the latter cells and is achieved by actin filament degradation correlated with the process of apoptosis (53) morphological changes were induced. Furthermore, co-inoculation of highly tumorigenic mouse mammalian tumor cells engineered to express MUC1 / Y protein and MUC1 / SEC expressing cells significantly reduced tumor growth of injected cells (67% Suggests that the presence of MUC1 / SEC within the microenvironment of injected highly tumorigenic mouse mammalian tumor cells reduces its tumorigenic potential.

  Table 1-Effect of anti-MUC1 mAb on MUC1 / SEC-MUC1 / Y interaction and cell death

ＭＵＣ１／ＳＥＣ−ＭＵＣ１／Ｙ相互作用に対する抗ＭＵＣ１ ｍＡｂの効果を、サンドイッチＥＬＩＳＡアッセイ（方法に記載される）によって分析した。

The effect of anti-MUC1 mAb on the MUC1 / SEC-MUC1 / Y interaction was analyzed by a sandwich ELISA assay (described in the method).

  (References)

The present invention will be understood and appreciated more fully from the following detailed description, taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows the expression of MUC1 protein in transfectants of epithelial cells. HBL-100 cells were transfected with either (A) an expression vector containing MUC1 / Y cDNA (H / Y2) or (B) a selection plasmid pSV2neo (H / N). Expression of various MUC1 isoforms was incubated with (A1, B1), anti-MUC1 / Y specific mAb BOS6E6 / 2, or (A2, B2) anti-tandem repeat mAb H23. Was analyzed by FIG. 2 shows the location of the different MUC1 protein isoforms and the epitope of the monoclonal specific antibody. FIGS. 3A-D show the effect of anti-MUC1 monoclonal antibody on the growth of H / Y2 cells using (A) MTT assay and (BD) by assessing cell morphology by microscopy. FIG. 4 shows that the effect of BOS7D10 on cell growth is dependent on serum concentration. H / Y2 cells were treated with 0.1% FCS (lane 6; 12), 0.2% FCS (lane 5; 11); 0.3% FCS (lane 4; 10), 1% FCS ( Lanes 3; 9); exposed to medium supplemented with 3% FCS (lanes 2; 8); and 10% FCS (lanes 1; 7). In order to determine the inhibitory effect of mAbs on cell proliferation, the effect of different mAbs on H / Y2 cells was analyzed by MTT. FIG. 5 shows the sequence of the MUC1 / REP protein. FIG. 6 shows the 59 amino acid sequence containing the epitope recognized by the BOS7D10 monoclonal antibody.

Claims (28)

  A method of inhibiting proliferation of a mammalian cell or growth of a mammalian cell, wherein the ligand requires an effective amount of the ligand that specifically binds to an epitope in the extracellular region of the transmembrane isoform of the MUC1 protein. Administering to a subject to selectively inhibit the proliferation of the mammalian cell or the growth of the mammalian cell.   A method for inducing mammalian cell death comprising administering to a subject in need thereof an effective amount of a ligand that specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein. And thereby selectively inducing the mammalian cell death.   A method of treating a subject having a disease with pathological proliferation of cells, wherein the ligand comprises an effective amount of the ligand that specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein. A method comprising administering to a subject in need thereof, thereby treating the disease.   A method of selectively inhibiting cell proliferation or cell growth, wherein an effective amount of a cell that expresses a MUC1 protein isoform specifically binds to an epitope in the extracellular region of the transmembrane isoform of the MUC1 protein. A method comprising the step of contacting with a ligand, thereby selectively inhibiting cell proliferation or cell growth.   A method of selectively inducing cell death, wherein a cell expressing a MUC1 protein isoform is contacted with an effective amount of a ligand that specifically binds to an eptop in the extracellular region of the transmembrane isoform of the MUC1 protein. A method comprising selectively inducing cell death.   The method according to claim 1, wherein the cell is a cancer cell.   The method of claim 6, wherein the cancer cell is an epithelial cancer cell.   8. The method of claim 7, wherein the epithelial cancer cell is a breast cancer cell, ovarian cancer cell, lung cancer cell or colon cancer cell.   6. The method according to claims 1-5, wherein the MUC1 isoform is MUC1 / Y, MUC1 / REP, or MUC1 / X.   6. The method according to claims 1-5, wherein the ligand is an antibody, peptide, antagonist or agonist.   6. The method according to claims 1-5, wherein the epitope is an amino acid sequence within the 59 amino acid sequence shown in SEQ ID NO: 1.   6. The method of claims 1-5, wherein the ligand is conjugated to a cytotoxic drug.   11. The method according to claim 10, wherein the antibody is a monoclonal antibody, a synthetic antibody, a polyclonal antibody or a chimera.   A method of treating a subject having a disease associated with pathological proliferation of cells, wherein an effective amount of the peptide comprising an amino acid sequence corresponding to the extracellular region of the transmembrane isoform of MUC1 protein is required. Administering to the subject to induce an increase in the level of antibodies specific for the peptide in the subject, thereby treating the disease.   15. A method according to claim 14, wherein the MUC1 isoform is MUC1 / Y, MUC1 / REP or MUC1 / X.   15. The method of claim 14, wherein the peptide comprises an amino acid sequence within the 59 amino acid sequence set forth in SEQ ID NO: 1.   An isolated antibody that specifically binds to an epitope in the extracellular region of the transmembrane isoform of the MUC1 protein.   18. The isolated antibody of claim 17, wherein the MUC1 protein isoform is a MUC1 / Y protein, a MUC1 / REP protein, or a MUC1 / X protein.   18. An isolated antibody according to claim 17, wherein the antibody is a monoclonal antibody, a synthetic antibody, a polyclonal antibody or a chimera.   18. The antibody according to claim 17, wherein the epitope is an amino acid sequence within the 59 amino acid sequence shown in SEQ ID NO: 1.   A hybridoma cell that produces a monoclonal antibody that binds to an epitope in the extracellular region of the transmembrane isoform of the MUC1 protein.   The hybridoma according to claim 21, wherein the isoform of the MUC1 protein is a MUC1 / Y protein, a MUC1 / REP protein or a MUC1 / X protein.   A pharmaceutical composition comprising an effective amount of a ligand that specifically binds to an epitope within the transmembrane isoform of the MUC1 protein and a pharmaceutically acceptable carrier.   24. The pharmaceutical composition of claim 23, wherein the ligand is conjugated to a cell killing agent.   24. The pharmaceutical composition of claim 23, wherein the MUC1 isoform is MUC1 / Y, MUC1 / REP or MUC1 / X.   24. The pharmaceutical composition of claim 23, wherein the epitope is an amino acid sequence within the 59 amino acid sequence set forth in SEQ ID NO: 1.   24. The pharmaceutical composition of claim 23, wherein the ligand is an antibody, peptide, antagonist or agonist.   28. The pharmaceutical composition of claim 27, wherein the antibody is a monoclonal antibody, a synthetic antibody, a polyclonal antibody or a chimera.

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